Structural mechanism of a DNA polymerase critical for developing antibiotic-resistance Abstract Antibiotic resistance is a dire and growing threat to human health, with over 20000 deaths per year occurring in the US alone due to drug-resistant pathogenic bacteria. Unfortunately, current antibiotics only target a few cellular pathways, leading to widespread emergence of multi-drug resistant strains. Moreover, development of new antibiotics has largely stalled. Here we propose investigation of DnaE2 as an exciting target for development of novel antibiotics that minimize evolution of drug resistance. DnaE2?s error-prone DNA polymerase activity is important for evolving drug resistance across a wide spectrum of bacteria. Therefore, a DnaE2 inhibitor could be used in combination therapy to prevent emergence of drug resistance. Furthermore, DnaE2 is critical for pathogenesis in Mycobacterium tuberculosis, but is not necessary for bacterial growth. Thus, DnaE2 inhibitors would prevent disease without selecting for drug-resistant bacterial strains. Finally, DnaE2 is unrelated to eukaryotic DNA polymerases, minimizing the chance of cross-reaction of DnaE2 inhibitors with human enzymes. These advantages make DnaE2 a particularly exciting new antibiotic target. Our expertise in structural, biophysical, and biochemical studies of DNA polymerases and other replication proteins uniquely positions the Kelch lab to investigate DnaE2?s potential as a drug target. We will determine the first atomic structure of DnaE2, which will reveal the molecular basis for DnaE2?s mutagenic activity as well as provide a blueprint for developing potent small molecule inhibitors. We will also measure the mutagenic preference for DnaE2, which will reveal how the enzyme drives the evolution of antibiotic resistance. Finally, we will screen for small molecule inhibitors of DnaE2, which will provide the foundation for developing novel ?anti-evolution? antibiotics.